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Molecular Medicine > Unit 2 - Cell Cycle > Flashcards

Unit 2 - Cell Cycle Flashcards

1
Q

key tasks for proliferating cells

A
  • replicate entire genome (ONCE) - fertilised egg → entire organism, cellular regeneration, in response to injury
  • separate duplicated chromosomes equally into daughter cells (UNLIKE stem and germ cells)
  • co-ordinate cell growth and proliferation by ensuring cells have enough energy and metabolites before entering into the processes
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2
Q

key events of the cell cycle

A
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3
Q

Gap phase G1

A

cell growth and regulatory events (checkpoints)

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4
Q

synthesis phase (S)

A

DNA replication occurs

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5
Q

gap phase 2 G2

A

cell growth and regulatory events (checkpoints)

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6
Q

M phase - mitosis

A

chromosomes are segregated and cells divide

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7
Q

when may cells exit cycle into G0

A

if terminally differentiated, senescent or if inhibitory signals are received

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8
Q

cell cycle timing

A

varies between cells depending on function and origin

Our genetic material is very large and will take a while to duplicate accurately, and segregate it between daughter cells

enzymes are conserved

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9
Q

DNA polymerases

A

highly conserved enzymes

require a 3’-OH group for activity and so require a primer

needed to extend DNA strands in a 5’ → 3’ direction

replicative polymerases = Pol δ and Pol ε

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10
Q

DNA replication overview

A

DNA is unwound and RNA primer molecules bound are synthesised by primase

primers are then extended by replicative polymerases (δ or ε) in a 5’ → 3’ direction

⇒ lagging strand is discontinuously synthesised

also leads to ‘end replication problem’ which is solved by telomerase

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11
Q

primase function

A

extend chains of DNA against the template provided by the pre-existing chromosome

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12
Q

Okazaki fragments

A

discontinuous segments being synthesised from RNA primer

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13
Q

telomerase function

A

allow intact replication of RNA ends

arises from the need of our polymerases to have our primer structure

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14
Q

steps in DNA synthesis - lagging strand

A
  1. helicase unwinds DNA, RPA loads - Pol α-primase synthesises a short primer
  2. Pol α displaced and Pol δ/Pol ε loaded
  3. Pol δ/Pol ε extend the primer
  4. downstream primer is removed (nuclease)
  5. Okazaki fragments are ligated

SIMILAR PROCESS OCCURS ON LEADING STRAND, ensuring both strands of DNA molecule are going to be duplicated completely

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15
Q

Pol α-primase function

A
  • Synthesise a short RNA primer against the template provided by the original DNA strand
  • Allow replicate of polymerases (Pol delta or epsilon) to extend in 5’ to 3’ direction from the 3’-OH in a process that will give rise to the new DNA strand
  • Primer is then removed by nuclease activty
  • Individual extended primers, now the actual DNA sequences, are ligated together again to give a continuous new DNA strand that will base pair with the original DNA strand and will be complementary to it due to extend of polymerase
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16
Q

M phase overview

A

chromosomes condense and attach to microtubules from mitotic spindle

all chromosomes must be attached before sister chromatids can separate

each daughter cell receives 1 set of chromosomes

chromosome segregation is irreversible so this process is highly-regulated

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17
Q

what happens when all chromatids have attached to the poles of mitotic spindle

A

the sister chromatids - duplicated pairs of chromosomes - will separate from one another

(held together after replication but prior to separation during mitosis)

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18
Q

6 phases of mitosis

A

prophase

prometaphase

metaphase

anaphase

telophase

cytokinesis

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19
Q

prophase

A

chromosomes begin to condense

requires condensin and DNA topoisomerase II

the (duplicated) centrosomes separate

histones undergo mitosis-specific modifications

Lose their diffused localised volume and begin to adopt a more tightly packed conformation that is mechanically necessary for them to migrate to opposite poles later in mitosis

intact nuclear envelope

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20
Q

what makes up chromatin

A

histones

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21
Q

prometaphase

A

microtubules from opposite spindle poles (centrosomes) bind chromosomes at kinetochores (waist) to initiate bipolar orientation

nuclear envelope breakdown occurs - can now spread out into the cell

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22
Q

metaphase

A

all chromosomes have made bipolar attachemnts to spindle poles

chromosomes align at metaphase plate

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23
Q

what is the key tightly-regulated step in mitosis

A

between metaphase and anaphase

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24
Q

anaphase

A

chromatids separate and move toward the opposite spindle poles - poles separate

nuclear envelope reassembly commences - reassembles around the chromosomes as they move apart

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25
Q

telophase

A

nuclear envelope reassembles around sister chromatids

poleward movement of chromosomes continues

cleavage plane is specified (Plane along which the cytoplasm will eventually separate)

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26
Q

cytokinesis

A

separation of daughter cells

formation of the cleavage furrow by a contractile ring of actin filaments (between the 2 masses of chromosomes)

chromosomes decondense and nuclear structures reform

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27
Q

vertebrate centromeres

A

the primary constriction in higher eukaryote chromosomes

heterochromatin region

centromeric heterochromatin carries the kinetochore

megabase arrays of highly repetitive satellite DNA

chromosomes contain varying amounts of satellite DNA of varying sequence

principal component of human CEN sequences is α-satellite DNA - monomer structure = 171 bp - arranged into complex repeats (2-32 monomers/repeat)

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28
Q

principal component of human CEN sequence

A

α-satellite DNA

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29
Q

key function of centromeric DNA sequence

A

it carries proteinic structure known as kinetochore

microtubules attach to kinetochore

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30
Q

vertebrate kinetochore

A

complex proteinaceous structure that controls chromosome-microtubule attachment and mitotic spindle assembly

trilaminar structure

kinetochore assembly on centromeric chromatin = temporally-regulated process involving several pathways

controls attachment of kinetochore to the microtubules

inner plate connects to chromosomes

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31
Q

key regulatory steps in cell cycle

A

making sure DNA replicates completely and once only - S phase control

ensuring DNA is intact before mitosis begins - G2 phase delays

making sure all chromosomes are segregated equally to 2 daughter cells - spindle checkpoint

overall co-ordination/timing

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32
Q

S phase control

A

pre-replicative complex binds to origins of replication (licensing) during late M/G1

licensing origins of replication are bound by initiator proteins (a large protein complex)

once activated, pre-replicative complex disassembles and cannot reassemble to reactivate an origin until the next cell cycle

each of the origins can fire only once per cell cycle

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33
Q

Orc1-6

A

Controlling the activation processes at the origins of replication

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34
Q

Cdc6 and Cdt1

A

Binds to origins of replication through out the chromosomes of the cell during late M phase/beginning of G1

Ensures the processes of replication can occur at each of the origins

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35
Q

Monitoring/regulating DNA integrity

A
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36
Q

centrosome

A

2 centrioles (barrels of microtubules composed of tubulin and centrin)

surrounded by cloud of pericentriolar material (PCM)

PCM contains γ-tubulin ring complex that nucleates microtubules

37
Q

contents of PCM

A

γ-tubulin ring complex that nucleates microtubules

38
Q

what do microtubules eventually connect to

A

kinetochore - forms on centromeric DNA

39
Q

what are centrioles

A

microtubule structures

contain tubulin in polymer and Ca2+ binding protein centrin

40
Q

centrosome duplication cycle

A

Ensures divisions organised by central poles are bipolar - pulling chromosomes in 2 directions

41
Q

mitotic spindle assembly

A

unstable centrosome microtubules are captured and stabilised by kinetochore binding until all are assembled

interpolar microtubule motors separate the spindle poles

bipolar orientation of chromosomes allows equatorial positioning

42
Q

spindle assembly/metaphase checkpoint

A
  • translation of absence of appropriate spindle-kinetochore interactions into a biochemical signal (wait anaphase) - If they lack microtubule attachments signal will block cell entry into anaphase
  • satisfaction of this checkpoint requires both occupancy of kinetochores by microtubules and inter-kinetochore tension
  • Sister kinetochores are attached to opposite poles of mitotic spindle - alleviates ‘wait anaphase’ signal and allow cell to progress
  • defective checkpoint signalling implicated in tumorigenesis
43
Q

control of cell cycle - what are the key regulators

how are they regulated

A

cyclin dependent kinases (CDKs)

cyclin levels oscillate during cell cycle

different cyclins are active at different stages in the cell cycle, being regulated through transcription and through degradation

key transcription regulators determine directionality of cell cycle e.g. pRb which controls E2F family transcription factors in G1/S, thus regulating cyclins A and E

CDK-cyclin activities are also regulated through phosphorylation

44
Q

oscillations of cyclins through a cell cycle

A

CDK activities are controlled by [regulated] cyclin levels

key transcriptional regulators determine cell cycle direction e.g. pRb, which controls E2F family transcription factors in G1/S, thus regulating cyclins A/E

(purple controlled through degradation - anaphase promoter complex)

45
Q

pRb

A

controls E2F family transcription factors in G1/S, thus regulating cyclins A/E

46
Q

cyclin/CDK combinations and activities

A
47
Q

Cdk1-cyclin B phosphorylation targets grouped on the basis of

A

protein function

48
Q

Cdk2-cyclin A phosphorylation targets grouped on the basis of protein function

A

activation of CDK cyclin pair determines the cell cycle stage

49
Q

key target sites in regulated activation of Cdk1

A

Active site cleft can be controlled by certain enzymes that can be phosphorylated in activating/repressing state

50
Q

feedback loops that control activation of Cdk1

A
51
Q

targeting of the cell cycle in cancer therapy

A
52
Q

anitmitotics

A

vincristine/vinblastine and taxol

impact proliferative status of cells - microtubules

53
Q

alkylating agents/Pt drugs

A

DNA damage

radiotherapy - Topo II inhibition (doxorubicin)

stop cells from entering into mitosis - perhaps cell death directly

54
Q

antimetabolites

A

block nucleic acid synthesis

impedes processes for successful S phase

55
Q

challenge of converting understanding of cell cycle into therapeutics

A

although CDKs are attractive targets, pan-CDK inhibitors are not established for use in the clinic, despite numerous trials

problems:

non-specificity of action (similarity of kinase catalytic sites), redundancy and unexpected side effects (involvement in other activities)

targeting other components of the cell cycle machinery has been more successful so far e.g. anti-replication drugs e.g. 5-FU/gemcitabine

56
Q

example of anti-replication drug

A

5-FU

gemcitabine

57
Q

3 mechanisms of DNA repair

A

base excision

nucleotide

mismatch

58
Q

activating point mutations in RAS - consequences

A

all compromise the GTPase activity of RAS

this prevents the hydrolysis of GTP on RAS, causing RAS to accumulate in the GTP-bound, active form

almost all RAS activation in tumours is accounted for by mutations in codons 12, 13 and 61

59
Q

Ras mutations in cancer

A

Mutations in a very small number of codons in human genome causes this

Only 3 bps

60
Q

codons involved in RAS activation

A

mutations in codons 12, 13 and 61

61
Q

causative DNA damage and cancer

A

major contributory factor

UV damage and skin cancer

cigarette smoke and lung cancer

62
Q

therapeutic DNA damage and cancer

A

major anti-cancer strategies kill tumour cells by DNA damage

chemotherapy e.g. cisplatin

radiotherapy

63
Q

sources of DNA damage

A

endogenous sources

free radicals

spontaneous base deamination/depurination

DNA replication errors (normal metabolism)

exogenous sources

radiation (ionising, UV)

chemicals (benzo[a]pyrene in cigarette smoke; aflatoxin; anti-cancer drugs)

64
Q

multiple pathways of DNA repair

A

different lesions are repaired through different repair mechanisms

65
Q

double-strand break results in

A

discontinuities in the sugar phosphate backbone of the double helix

66
Q

base excision repair

A

DNA base damage occurs continuously in our cells

generation of abasic (apurinic/apyrimidinic ⇒ AP) sites through hydrolytic cleavage of N-glycosidic bond - 2,000-10,000/cell/day

67
Q

cytosine → uracil

A

100-500/cell/day

68
Q

adenine → hypoxanthine

A

10-50/cell/day

69
Q

oxidative damage example

A

8-oxo-deoxyguanine (8-oxo-dG)

100-500/cell/day

70
Q

alkylation damage example

A

O6-methylguanine

71
Q

what do deamination changes cause

A

inappropriate base pairing, which is mutagenic

72
Q

mechanism of BER

A

DNA glycosylase recognises damaged base and removes it, leaving abasic (AP) site

AP endonuclease cleaves the DNA at this AP site

DNA polymerase carries out repair synthesis

DNA ligase rejoins sugar-phosphate backbone

73
Q

nucleotide excision repair

A

removes DNA lesions that strongly distort DNA structure

especially UV-induced lesions

beach in strong sunlight - 40,000 damaged sites per hour in exposed epidermal (skin) cell

due to UV light (200-320 nm)

most UV-C light (100-290 nm) is absorbed by ozone layer and air will absorb UV to 200 nm

74
Q

xeroderma pigmentosum

A

lack of DNA repair of UV-damafe results in skin cancer susceptibility

XP is a rare human disease caused by inherited mutations in XP genes

leads to extreme susceptibility to skin cnacer (melanoma, squamous cell carcinoma) arising from solar UV-induced DNA damage

75
Q

NER mechanism

A

DNA distortion from the damaged bases is recognised by a complex containing XPA and XPC (recognise the damaged DNA)

in transcribed DNA, the stalled RNA polymerase can act as a recognition signal

DNA helix is unwound by XPB/XPD

XPF/Ercc I and XPG nick the DNA 5’ and 3’ of the lesion

DNA polymerase δ or ε synthesises the excised sequence and DNA ligase seals the nicks

76
Q

mismatch repair

A

human genome - 3 x 109 bps

genome must be accurately replicated (during S phase) each time a cell divides

occasionally DNA polymerase (particularly pol δ) makes an error in copying DNA

mismatch repair required to correct these errors

77
Q

mechanism of MMR

A

mismatch recognition proteins (MSH2/MSH6) detect the error

a sliding clamp is formed to find a single-strand nick (newly synthesised DNA)

DNA is exonucleolytically degraded until the mismatch

repair synthesis is performed by DNA polymerase δ or ε and DNA ligase seals the nicks

78
Q

MMR and cancer

A

defective MMR causes microsatellite instability

hereditary non-polyposis colon cancer results from germline mutations in MSH2 and MLH1

e.g. 9/11 HNPCC cell lines studied had a mutation in a stretch of the type II TGF-β receptor sequence

this causes the instability of the (truncated) receptor, making cells insensitive to the growth inhibition signals of TGF-β

79
Q

DNA double-strand breaks

A

discontinuities in both strands of the DNA double helix

particularly hazardous to cells because of the risk of translocations of the loss of genetic material

TELOMERE = a special protective nucleoprotein complex at the ends of linear chromosomes to ensure that they are not treated as broken ends

80
Q

telomere

A

a special protective nucleoprotein complex at the ends of linear chromosomes to ensure that they are not treated as broken ends

81
Q

sources of DSBs

A

ionising radiation (X rays, gamma rays) - short wavelength, high energy

generates free radicals in cells

leads to DNA damage - single-strand breaks (ssbs) and double-strand breaks (dsbs) in DNA

carcinogenic

leads to cell death ⇒ used in radiotherapy

localised doses can be very high (50Gy), but > 5Gy whole-body irradiation is lethal

radiomimetic chemotherapeutic drugs (topoisomerase II inhibitors e.g. doxorubicin/adriamycin)

repair of single-strand breaks - rapid in cells - requires poly (ADP-ribose) polymerase (PARP)

82
Q

examples of topoisomerase II inhibitors

A

doxorubicin

adriamycin

83
Q

non-homologous end-joining

A

repairs DSB with no requirement for major homology - potentially mutagenic

principle DSB repair mechanism in mammalian cells

breaks are bound by Ku70/Ku80 dimer to initiate repair

broken ends are religated by DNA ligase IV/XRCC4

this process is critical in V(D)J recombination during immune system development

84
Q

what is lack of sequence homology useful in

A

allowing us to have a large immune repertoire

85
Q

homologous recombination

A

accurate, template-directed repair mechanism - with use of intact sister chromatid available - Limited to post-replicational phase - needs intact alternative template for repair

initiated by resection at break to expose a tract of ssDNA

Rad5 I recombinase forms a nucleoprotein filament on ssDNA which carries out a homology search

strand invasion allows templated DNA synthesis and the resulting Holliday junction is cleaved to yield repaired sequence

BRCA2 is a key component in HR

86
Q

DNA damage responses and disease

A

germline DNA repair defects in disease (cancer predisposition and other non-cancer diseases)

spontaneous mutations arising in carcinogenesis

exploitation of DNA repair pathways to kill tumour cells

87
Q

germline DNA repair gene defects predisposing to cancer

A
88
Q

DNA repair defects differ among cancer types

A
89
Q

exploiting our understanding of DNA repair

A

synthetic lethality

spontaneous breaks that occur during normal DNA replication are repaired by HR and Parp-I dependent ssb repair (Normally available to cell - if spontaneous break occurs EITHER OR)

BRCA2-defective cells have defective HR so rely on Parp I to repair such breaks

inhibition of Parp I in BRCA2-negative cells is a specific and potent killing mechanism

BRCA2 cells do not have homologous recombination

Rely completely on PARP I

Inhibition of this pathway makes the spontaneous lesions that occur during normal replication lethal to the cells

Molecular Medicine (7 decks)

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